CN104450657A - Nitrile hydratase as well as encoding gene and application thereof - Google Patents
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Abstract
The invention discloses a nitrile hydratase as well as an encoding gene and application thereof. The nitrile hydratase is prepared from an alpha subunit and a beta subunit, wherein an amino acid sequence of the alpha subunit is shown as SEQ ID NO.4, and an amino acid sequence of the beta subunit is shown as SEQ ID NO.5. According to the nitrile hydratase as well as the encoding gene and the application thereof, a nitrile hydratase gene is cloned from acid-producing Klebsiella spp KCTC 1686, and the nitrile hydratase with high expression level, high activity, wide substrate spectrum and chiral selectivity is successfully obtained after the gene is expressed.
Description
Technical field
The present invention relates to genetically engineered field, particularly relate to a kind of Nitrile hydratase and encoding gene thereof and application.
Background technology
Nitrile is the important compound of a class, and its hydrolysis reaction is widely used in the synthesis of amino acid, acid amides, carboxylic acid and derivative thereof, occupies and consequence in organic synthesis.The method of nitrile hydrolysis mainly contains chemical hydrolysis and biotransformation method, and to have mild condition, environmental pollution little and can realize the advantages such as chemical, region and enantio-selectivity for biotransformation method compared with chemical hydrolysis.Biotransformation method relates generally to Nitrile hydratase, Ntn hydrolase and nitrilase.Wherein Nitrile hydratase the hydration of catalysis nitrile can generate acid amides, and acid amides catalytic hydrolysis is generated carboxylic acid cpd by Ntn hydrolase further, and nitrilase directly can generate amide compound by catalysis nitrile.
The distribution of nitrile hydratase production microorganism is quite widely, as rhodococcus, pseudomonas, Nocardia bacteria, Selective medium, Alcaligenes, coryneform bacteria etc.In the suitability for industrialized production of some of them microorganism for acrylamide, niacinamide, as the patent No. be US007405064B2 patent discloses a kind of wild Comamonas testosteroni 5-MGAM-4D; Patent No. ZL88106735 patent discloses a kind of wild rose look rhodococcus J-1; The patent No. be ZL86100062 patent discloses a kind of rhodococcus S-6, oxidation Arthrobacter and microbacterium flavum; The patent No. be ZL99106291.4 patent discloses a kind of wild thermophilic Selective medium JCM3095; The patent No. be ZL03115536.7 patent discloses a kind of wild propionic acid bar bacterium.
At present, the method for Production of Acrylamide by Microbial Method, niacinamide is utilized mainly to carry out the work from the aspect such as foundation and transformation of the screening of bacterial strain and domestication and production technique.But, wherein the enzyme of ubiquity wild-type strain is lived the problems such as poor stability, not high to the tolerance of substrate and product, quality product is stable not, simultaneously in wild strain except Nitrile hydratase, also there is Ntn hydrolase and nitrilase, it can cause by product nicotinic acid and acrylic acid generation, thus have a strong impact on the seed output and quality of niacinamide and acrylamide, increase difficulty and the production cost of separation and purification simultaneously.
Along with developing rapidly of biotechnology, people recognize there is the engineering strain of nitrile hydratase activity by genetic engineering technique means can with solving an above-mentioned difficult problem.The advantage that Nitrile hydratase has many wild mushrooms not have is expressed with genetic engineering bacterium, as can clonal expression Nitrile hydratase separately, block the generation of side reaction in catalytic reaction process, can not make acid amides while generation, have again part to be degraded, thus improve the seed output and quality of product; The strong adaptability of genetic engineering bacterium, fermentation period are short in addition, are conducive to realizing large scale culturing and suitability for industrialized production.Current, the research of restructuring Nitrile hydratase has caused people to pay attention to widely, through numerous researcher make great efforts for many years also achieve considerable achievement, but also there are problems, expression level as enzyme is low, thermostability is not high, poor to the tolerance of substrate and product, this seriously constrains the industrial applications of Nitrile hydratase.
Summary of the invention
The invention provides a kind of high expression level amount, high reactivity and substrate spectrum is wide, the Nitrile hydratase of tool chiral selectivity.
A kind of Nitrile hydratase, be made up of α subunit and β subunit, the aminoacid sequence of described α subunit is as shown in SEQ ID NO.4, and the aminoacid sequence of β subunit is as shown in SEQ ID NO.5.The called after NHaseK of this Nitrile hydratase.
Present invention also offers a kind of gene of described Nitrile hydratase of encoding.
Preferably, described gene comprises the gene of the gene of the coding for alpha subunit of base sequence as shown in SEQ ID NO.1 and the coding β subunit as shown in SEQ IDNO.2.This base sequence derives from Klebsiella oxytoca KCTC 1686 (Klebsiellaoxytoca KCTC 1686), by 1278 based compositions.From the 1st, 5 ' end to 609 bit base encoding nitrile hydratase α subunits; From the 625th, 5 ' end to 1278 bit base encoding nitrile hydratase β subunits.Further, base sequence is as shown in SEQ ID NO.7.
Described gene also comprises the base sequence that encoding nitrile hydratase activates son.Described Nitrile hydratase activates the base sequence of son as shown in SEQ ID NO.3.For improving expression activity, described gene is connected with the gene (SEQ IDNO.3) activating son after modifying.Further, base sequence is as shown in SEQ ID NO.8.
The invention also discloses a kind of Nitrile hydratase and activate son, aminoacid sequence is as shown in SEQ ID NO.6.
The invention also discloses a kind of expression cassette of arbitrary described gene, recombinant vectors and transformant.
The present invention again discloses a kind of described Nitrile hydratase and is preparing the application in amide compound.
Due to the singularity of aminoacid sequence; the fragment of any peptide protein containing aminoacid sequence shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or its variant; as long as the fragment of this peptide protein or peptide protein variant and aforementioned amino acid sequences homology are more than 90%; and there is identical enzyme activity; all can realize the object of the invention, belong to the row of scope.
Due to the singularity of nucleotide sequence; any variant containing polynucleotide shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3; as long as itself and this polynucleotide have more than 90% homology, all can realize the object of the invention, belong to the row of scope.
The invention provides a kind of recombinant vectors and the transformant that comprise described nitrile hydratase gene.
Present invention also offers a kind of described recombinant vectors and transformant transforms the genetic engineering bacterium obtained.
Invention further provides the application of a kind of described Nitrile hydratase in catalysis nitrile compound generation acid amides.
Described nitrile compound is that shown in formula (I) and (II), nitrile compound generates the application in corresponding acid amides:
In formula (I) ~ (II):
X:OH, H, NH
2, alkyl;
R:H, optionally by NH
2replace, branch or unbranched, that there is 1-12 C atom saturated alkyl; Unsaturated alkyl molecule or unbranched, there is double bond and 1-12 C atom; There is the cycloalkyl of 3-6 C atom;
R ': H, there is the alkyl of 1-3 C atom;
R ": the unsaturated ring of monokaryon or double-core, it has 6-12 C atom, and it is optionally replaced by 1 or 2 Cl, Br, F; There is the alkyl nitrile group of 1-6 C atom.
The present invention is cloned into nitrile hydratase gene from Klebsiella oxytoca KCTC 1686, successfully obtains to have high expression level amount and high reactivity and substrate spectrum is wide, the Nitrile hydratase of tool chiral selectivity after this genetic expression.
Accompanying drawing explanation
Fig. 1 is the pcr amplification electrophorogram of nitrile hydratase gene of the present invention;
M: nucleic acid Marker; 1 and 2: the pcr amplification product of Nitrile hydratase.
Fig. 2 is the collection of illustrative plates of recombinant plasmid pET-30a (+)-NHaseK of the present invention.
Fig. 3 is the SDS-PAGE electrophorogram of engineering strain E.coli BL21 (DE3) of the present invention/pET-30a (+)-NHaseK abduction delivering product.
M: lower molecular weight standard protein; The broken cytosol of 1:pET-30a (+) empty plasmid contrast;
After 2: genetically engineered E.coli BL21 (DE3)/pET-30a (+)-NHaseK induces, thalline breaks cytosol;
3: genetically engineered E.coli BL21 (DE3)/pET-30a (+)-NHaseK induces thalline to break born of the same parents' supernatant liquor;
4: genetically engineered E.coli BL21 (DE3)/pET-30a (+)-NHaseK induces thalline to break born of the same parents' precipitation, and arrow indicates α subunit, β subunit respectively and activates the position of sub-P17K.
Fig. 4 is the structure schematic diagram of expression plasmid pET-30a (+)-NHaseK.
Embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited only to this.
Materials and methods in embodiment is as follows:
Experimental technique in the present invention is ordinary method if no special instructions, " the Molecular Cloning: A Laboratory guide " that specifically can write see J. Pehanorm Brooker etc.
Restriction enzyme EcoRI, HindIII and T4 DNA ligase used in the embodiment of the present invention purchased from TaKaRa, precious biotechnology (Dalian) company limited; Genome extracts test kit, plasmid extraction kit, DNA recovery purification kit purchased from Axygen Hangzhou company limited; E.coli DH5 α, E.coli BL21 (DE3), plasmid pET-30a (+) are purchased from Novagen company; DNA marker, FastPfu archaeal dna polymerase, lower molecular weight standard protein, agarose electrophoresis reagent are purchased from Beijing Quanshijin Biotechnology Co., Ltd; Primer synthesis is completed by Shanghai Sheng Gong biotechnology company limited with sequence work.Above reagent using method is with reference to catalogue.The Klebsiella oxytoca KCTC 1686 (Klebsiellaoxytoca KCTC 1686) that the present invention adopts is purchased from Korea S's Type Culture Collection (Korean Collection for Type Cultures (KCTC))
Embodiment 1
One, clone from Klebsiella oxytoca KCTC 1686 (Klebsiella oxytoca KCTC 1686) genome Nitrile hydratase and its activate subbase because of
Primer NH-F and NH-R is designed according to Klebsiella oxytoca KCTC 1686 genomic dna sequence (GenBank accession number: CP003218.1).
NH-F sequence: 5 '-CCG
gAATTCaTGAGCCATAAACACGACCACG-3 '
NH-R sequence: 5 '-TTCCC
aAGCTTgTTATGGTGTAACTCCATTATCG-3
Restriction enzyme site EcoRI, HindIII (shown in underscore) is added respectively in the primer of upstream and downstream.With Klebsiellaoxytoca KCTC 1686 genomic dna for template, NH-F and NH-R is that primer carries out pcr amplification, PCR reaction system and reaction conditions as follows:
PCR amplification system:
Pcr amplification condition:
1) denaturation: 95 DEG C of 5min;
2) sex change: 98 DEG C of 10s; Annealing: 57 DEG C of 15s; Extend: 72 DEG C of 60s; Circulate 30 times altogether;
3) extend: 72 DEG C of 10min;
4) 2.0h is preserved for 4 DEG C.
Detect pcr amplification product with 0.8% agarose gel electrophoresis, product is single band, and size is about 1700bp (as shown in Figure 1).Reclaim purification kit with DNA and carry out purifying recovery to pcr amplification product, concrete steps are with reference to this test kit specification sheets.
Two, the structure of expression vector and engineering bacteria
Object fragment after first being reclaimed by purifying and pET-30a (+) empty plasmid of extraction carry out double digestion with restriction enzyme EcoRI and HindIII respectively.Reclaim purification kit with DNA afterwards to carry out purifying to digestion products and reclaim the Nucleotide small segment scaled off to remove restriction enzyme and enzyme.Finally object fragment be connected with pET-30a (+) plasmid with T4 DNA ligase, linked system is as shown in table 1 below:
Table 1 pET-30a (+)-NHaseK recombinant expression plasmid linked system
Above-mentioned each reagent is mixed gently, and is put in 16 DEG C of metal baths and connects 12h.Afterwards with connecting product conversion E.coliDH5a competent cell, be coated with dull and stereotyped, choose single bacterium colony and carry out LB liquid culture, PCR method identifies the positive transformant successfully constructed.From E.coli DH5a positive transformants bacterial strain, extract recombinant plasmid pET-30a (+)-NHaseK with Axygen plasmid extraction kit, and transform expressive host E coli BL21 (DE3) competent cell with it.Verify the recon of conversion by PCR method, verify errorless after genetic engineering bacterium be E.coli BL21 (DE3)/pET-30a (+)-NHaseK.
Three, the expression of restructuring Nitrile hydratase
Genetically engineered E.coli BL21 (DE3)/pET-30a (+)-NHaseK built is connected to 5mL containing in the LB liquid nutrient medium of 50 μ g/ml Kan, 37 DEG C of concussion overnight incubation.Get 1mL nutrient solution and be forwarded to 50mL equally containing in the fresh LB liquid nutrient medium of 50 μ g/ml Kan, 37 DEG C of concussions are cultured to OD
600when reaching about 0.8, adding IPTG to its final concentration is 0.5mM, induces 18h at 20 DEG C.The SDS-PAGE electrophorogram of its abduction delivering product, as shown in Figure 3.
Embodiment 2 genetic engineering bacterium catalyzing propone nitrile generates acrylamide
Enzyme activity unit is defined as: at reaction conditions, the enzyme amount of per minute catalytic substrate reaction generation 1 μm of ol product.
Get the fermented liquid of 25ml embodiment 1 engineering bacteria E.coli BL21 (DE3)/pET-30a (+)-NHaseK, 10000rpm, 10min collected by centrifugation thalline, then use the resuspended somatic cells of 250ml 50mM Tris-HCl (pH 7.5) damping fluid, obtain the resting cell suspension of engineering bacteria E.coli BL21 (DE3)/pET-30a (+)-NHaseK.In re-suspension liquid, add 1.5ml vinyl cyanide, at 35 DEG C, carry out hydration reaction, react 2 hours.Use the content of vinyl cyanide in vapor-phase chromatography detection reaction system and acrylamide afterwards.To found that in reaction system without acrylonitrile residue, be all converted into acrylamide.
Embodiment 3 genetic engineering bacterium catalysis butyronitrile generates butyramide
Get the fermented liquid of 25ml embodiment 1 engineering bacteria E.coli BL21 (DE3)/pET-30a (+)-NHaseK, 10000rpm, 10min collected by centrifugation thalline, then use the resuspended somatic cells of 250ml 50mM Tris-HCl (pH 7.0) damping fluid, obtain the resting cell suspension of engineering bacteria E.coli BL21 (DE3)/pET-30a (+)-NHaseK.In re-suspension liquid, add 7.0g butyronitrile, at 35 DEG C, carry out hydration reaction, react 2 hours.Use the content of butyronitrile in vapor-phase chromatography detection reaction system and butyramide afterwards.Found that in reaction system and remain without butyronitrile, be all converted into butyramide.
Embodiment 4 genetic engineering bacterium catalysis methacrylonitrile generates Methacrylamide
Get the fermented liquid of 25ml embodiment 1 engineering bacteria E.coli BL21 (DE3)/pET-30a (+)-NHaseK, 10000rpm, 10min collected by centrifugation thalline, then use the resuspended somatic cells of 250ml 50mM Tris-HCl (pH 7.0) damping fluid, obtain the resting cell suspension of engineering bacteria E.coli BL21 (DE3)/pET-30a (+)-NHaseK.In re-suspension liquid, add 3.0g methacrylonitrile, at 30 DEG C, carry out hydration reaction, react 2 hours.Use the content of methacrylonitrile in vapor-phase chromatography detection reaction system and Methacrylamide afterwards.Found that in reaction system and remain without methacrylonitrile, be all converted into Methacrylamide.
Embodiment 5 genetic engineering bacterium catalysis nicotinonitrile generates niacinamide
Get the fermented liquid of 25ml embodiment 1 engineering bacteria E.coli BL21 (DE3)/pET-30a (+)-NHaseK, 10000rpm, 10min collected by centrifugation thalline, then use the resuspended somatic cells of 250ml 50mM Tris-HCl (pH 7.0) damping fluid, obtain the resting cell suspension of engineering bacteria E.coli BL21 (DE3)/pET-30a (+)-NHaseK.In re-suspension liquid, add 5.0g nicotinonitrile, at 35 DEG C, carry out hydration reaction, react 2 hours.Use the content of nicotinonitrile in high performance liquid chromatography detection reaction system and niacinamide afterwards.Found that in reaction system and remain without nicotinonitrile, be all converted into niacinamide.High performance liquid chromatograph used is Agilent 1100, and chromatographic column is C
18post (5 μm, 4.6 × 150mm).
Embodiment 6 genetic engineering bacterium catalysis cyanobenzene generates benzamide
Get the fermented liquid of 25ml embodiment 1 engineering bacteria E.coli BL21 (DE3)/pET-30a (+)-NHaseK, 10000rpm, 10min collected by centrifugation thalline, then use the resuspended somatic cells of 250ml 50mM Tris-HCl (pH 7.0) damping fluid, obtain the resting cell suspension of engineering bacteria E.coli BL21 (DE3)/pET-30a (+)-NHaseK.In re-suspension liquid, add 3.5g cyanobenzene, at 35 DEG C, carry out hydration reaction, react 2 hours.Use the content of cyanobenzene in high performance liquid chromatography detection reaction system and benzamide afterwards.Found that in reaction system and remain without cyanobenzene, be all converted into benzamide.High performance liquid chromatograph used is Agilent 1100, and chromatographic column is C
18post (5 μm, 4.6 × 150mm).
Embodiment 7 genetic engineering bacterium catalysis benzyl cyanide generates phenylacetamide
Get the fermented liquid of 25ml embodiment 1 engineering bacteria E.coli BL21 (DE3)/pET-30a (+)-NHaseK, 10000rpm, 10min collected by centrifugation thalline, then use the resuspended somatic cells of 250ml 50mM Tris-HCl (pH 7.0) damping fluid, obtain the resting cell suspension of engineering bacteria E.coli BL21 (DE3)/pET-30a (+)-NHaseK.In re-suspension liquid, add 3.0g benzyl cyanide, at 35 DEG C, carry out hydration reaction, react 2 hours.Use the content of benzyl cyanide in high performance liquid chromatography detection reaction system and phenylacetamide afterwards.Found that in reaction system and remain without benzyl cyanide, be all converted into phenylacetamide.High performance liquid chromatograph used is Agilent 1100, and chromatographic column is C
18post (5 μm, 4.6 × 150mm).
Embodiment 8 genetic engineering bacterium catalysis Alpha-Methyl benzyl cyanide generates Alpha-Methyl phenylacetamide
Get the fermented liquid of 25ml embodiment 1 engineering bacteria E.coli BL21 (DE3)/pET-30a (+)-NHaseK, 10000rpm, 10min collected by centrifugation thalline, then use the resuspended somatic cells of 250ml 50mM Tris-HCl (pH 7.0) damping fluid, obtain the resting cell suspension of engineering bacteria E.coli BL21 (DE3)/pET-30a (+)-NHaseK.In re-suspension liquid, add 2.5g Alpha-Methyl benzyl cyanide, at 35 DEG C, carry out hydration reaction, reaction 30min.Content and the optical purity thereof of Alpha-Methyl benzyl cyanide in detection reaction system and Alpha-Methyl phenylacetamide is come afterwards by high performance liquid chromatography.Found that genetic engineering bacterium is when catalysis racemic ' alpha '-methylbenzeneacetonitrile, shows S-isomer stereoselectivity.High performance liquid chromatograph used is Agilent 1100, and chromatographic column is AY-RH chiral column (5 μm, 4.6 × 150mm, CHIRALPAK).
Embodiment 9 genetic engineering bacterium catalysis 2,2-dimethyl cyclopropylniitrile generates 2,2-diformazan basic ring propionic acid amide
Get the fermented liquid of 25ml embodiment 1 engineering bacteria E.coli BL21 (DE3)/pET-30a (+)-NHaseK, 10000rpm, 10min collected by centrifugation thalline, then use the resuspended somatic cells of 250ml 50mM Tris-HCl (pH 7.0) damping fluid, obtain the resting cell suspension of engineering bacteria E.coli BL21 (DE3)/pET-30a (+)-NHaseK.In re-suspension liquid, add 1.5g 2,2-dimethyl cyclopropylniitrile, at 35 DEG C, carry out hydration reaction, reaction 30min.Content and the optical purity thereof of 2,2-dimethyl cyclopropylniitriles in detection reaction system and 2,2-diformazan basic ring propionic acid amide is come afterwards by high performance liquid chromatography.Found that genetic engineering bacterium is when catalysis racemize 2,2-dimethyl cyclopropylniitrile, shows S-isomer stereoselectivity.High performance liquid chromatograph used is Agilent 1100, and chromatographic column is AY-RH chiral column (5 μm, 4.6 × 150mm, CHIRALPAK).
Embodiment 10 genetic engineering bacterium catalysis 2-(4-chloro-phenyl-)-3-methylbutyronitrile generates 2-(4-chloro-phenyl-)-3-methylbutyryl amine
Get the fermented liquid of 25ml embodiment 1 engineering bacteria E.coli BL21 (DE3)/pET-30a (+)-NHaseK, 10000rpm, 10min collected by centrifugation thalline, then use the resuspended somatic cells of 250ml 50mM Tris-HCl (pH 7.0) damping fluid, obtain the resting cell suspension of engineering bacteria E.coli BL21 (DE3)/pET-30a (+)-NHaseK.In re-suspension liquid, add 1.0g 2-(4-chloro-phenyl-)-3-methylbutyronitrile, at 35 DEG C, carry out hydration reaction, reaction 30min.Content and the optical purity thereof of 2-(4-chloro-phenyl-)-3-methylbutyronitrile in detection reaction system and 2-(4-chloro-phenyl-)-3-methylbutyryl amine is come afterwards by high performance liquid chromatography.Found that genetic engineering bacterium is when catalysis racemize 2-(4-chloro-phenyl-)-3-methylbutyronitrile, shows S-isomer stereoselectivity.High performance liquid chromatograph used is Agilent 1100, and chromatographic column is AY-RH chiral column (5 μm, 4.6 × 150mm, CHIRALPAK).
Comparative example 1
Fallon, R.d. (Applied Microbiology and Biotechnology, 47,156-161,1997) hydration reaction of catalysis 2-(4-chloro-phenyl-)-3-methylbutyronitrile etc. is carried out with pseudomonas putida NRRL-18668 (Pseudomonas putida NRRL-18668).Find that Nitrile hydratase energy catalysis 2-(4-the chloro-phenyl-)-3-methylbutyronitrile hydration that this wild mushroom contains generates 2-(4-chloro-phenyl-)-3-methylbutyryl amine, and show (S) type stereoselectivity, enzyme activity is 7.1 × 10
-3μm ol/min/mg wet cell.Its optimal reactive temperature is 30 DEG C, and when temperature is more than 30 DEG C, zymoprotein starts rapid deactivation.During engineering bacteria E.coliBL21 (DE3) of the present invention/pET-30a (+)-NHaseK catalysis 2-(4-chloro-phenyl-)-3-methylbutyronitrile, its enzyme activity reaches 40.0 × 10
-3μm ol/min/mg wet cell.Optimal reactive temperature is 35 DEG C, and below 40 DEG C, zymoprotein shows goodish stability.
Comparative example 2
Shun-Ichi Masutomo (Bioscience Biotechnology and Biochemistry, 59 (4), 720-722,1995) hydration reaction of catalysis 2-(4-chloro-phenyl-)-3-methylbutyronitrile etc. is carried out with pseudomonas B21C9 (pseudomonas sp.B21C9).Find that Nitrile hydratase energy catalysis 2-(4-the chloro-phenyl-)-3-methylbutyronitrile hydration that this wild mushroom contains generates 2-(4-chloro-phenyl-)-3-methylbutyryl amine, and show certain (S) type stereoselectivity, but its activity is lower, is only 1.8 × 10
-4μm ol/min/mg stem cell.During engineering bacteria E.coli BL21 (DE3) of the present invention/pET-30a (+)-NHaseK catalysis 2-(4-chloro-phenyl-)-3-methylbutyronitrile, its enzyme activity reaches 7.8 × 10
-2μm ol/min/mg stem cell.
Claims (10)
1. a Nitrile hydratase, is made up of α subunit and β subunit, it is characterized in that, the aminoacid sequence of described α subunit is as shown in SEQ ID NO.4, and the aminoacid sequence of β subunit is as shown in SEQ ID NO.5.
2. the gene of a coding Nitrile hydratase according to claim 1.
3. gene as claimed in claim 2, is characterized in that, comprise the sequence of the sequence of the coding for alpha subunit of base sequence as shown in SEQ ID NO.1 and the coding β subunit as shown in SEQ ID NO.2.
4. gene as claimed in claim 3, it is characterized in that, base sequence is as shown in SEQ ID NO.7.
5. gene as claimed in claim 2, is characterized in that, also comprises the sequence that encoding nitrile hydratase activates son.
6. gene as claimed in claim 5, is characterized in that, described Nitrile hydratase activates the base sequence of son as shown in SEQ IDNO.3.
7. gene as claimed in claim 6, it is characterized in that, base sequence is as shown in SEQ ID NO.8.
8. a Nitrile hydratase activates son, and it is characterized in that, aminoacid sequence is as shown in SEQ ID NO.6.
9. the expression cassette comprising the arbitrary described gene of claim 2 ~ 7, recombinant vectors and transformant.
10. Nitrile hydratase as claimed in claim 1 generates the application in acid amides at catalysis nitrile compound.
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| CN104774829A (en) * | 2015-04-22 | 2015-07-15 | 江南大学 | Fusion type nitrile hydratase with improved specific enzyme activity and stability |
| CN107881163A (en) * | 2017-09-29 | 2018-04-06 | 浙江大学 | A kind of heat-resisting nitrile hydratase, engineering bacteria and its application in catalysis nitrile compound hydration reaction generation acid amides |
| CN107916271A (en) * | 2017-09-29 | 2018-04-17 | 浙江大学 | A kind of high-efficiency expression method for recombinating nitrile hydratase |
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| CN112322607A (en) * | 2020-11-20 | 2021-02-05 | 江南大学 | Fusion type nitrile hydratase and application thereof |
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| US20230159452A1 (en) * | 2020-04-24 | 2023-05-25 | Pharmazell Gmbh | Regioselective oxidation of heterocyclic alpha-amino amides |
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| US20040225116A1 (en) * | 2003-05-08 | 2004-11-11 | Payne Mark S. | Nucleic acid fragments encoding nitrile hydratase and amidase enzymes from comamonas testosteroni 5-MGAM-4D and recombinant organisms expressing those enzymes useful for the production of amides and acids |
| CN1584024A (en) * | 2004-05-24 | 2005-02-23 | 清华大学 | Nitrile hydratase and its coding gene and use |
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| US20040225116A1 (en) * | 2003-05-08 | 2004-11-11 | Payne Mark S. | Nucleic acid fragments encoding nitrile hydratase and amidase enzymes from comamonas testosteroni 5-MGAM-4D and recombinant organisms expressing those enzymes useful for the production of amides and acids |
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Cited By (7)
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| CN104774829A (en) * | 2015-04-22 | 2015-07-15 | 江南大学 | Fusion type nitrile hydratase with improved specific enzyme activity and stability |
| CN104774829B (en) * | 2015-04-22 | 2017-10-31 | 江南大学 | A kind of specific enzyme activity and stability-enhanced pattern of fusion nitrile hydratase |
| CN107881163A (en) * | 2017-09-29 | 2018-04-06 | 浙江大学 | A kind of heat-resisting nitrile hydratase, engineering bacteria and its application in catalysis nitrile compound hydration reaction generation acid amides |
| CN107916271A (en) * | 2017-09-29 | 2018-04-17 | 浙江大学 | A kind of high-efficiency expression method for recombinating nitrile hydratase |
| WO2021018170A1 (en) * | 2019-08-01 | 2021-02-04 | 安徽瑞邦生物科技有限公司 | Preparation of transformed microorganism for producing pyridine carboxamide with low by-product and use thereof |
| CN112322607A (en) * | 2020-11-20 | 2021-02-05 | 江南大学 | Fusion type nitrile hydratase and application thereof |
| CN112322607B (en) * | 2020-11-20 | 2023-03-28 | 江南大学 | Fusion type nitrile hydratase and application thereof |
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